Ractopamine is a β-adrenoreceptor agonist that excites sympathetic nerves. It has been used to increase cattle weight, for breeding, and to enhance muscle content in some countries. The concentration allowed in the environment is below 10 parts per billion (ppb). However, there are increasing concerns about the effect of long-term ractopamine on health. Our study aimed to investigate the potential effects of a “safe” dosage of ractopamine on urinary tract by using genitourinary cell cultures and our well-established translational model, Drosophila melanogaster. The results showed that ractopamine dose-dependently induces cytotoxicity in SV40 MES 13 and SV-HUC-1 cells. After 21 days of 10 ppb ractopamine administration, the rate of crystal formation in the ractopamine group significantly increased. We also found that long-term administration of ractopamine to flies decreases their climbing ability and shortens their lifespan. Overall, the long-term effects of ractopamine on the urinary tract system were evident in our cell and animal studies. In particular, renal mesangial and urothelial cells are more susceptible to damage; urolithiasis and neurological damage are other possible side effects of ractopamine. These effects on the human urinary tract should be further investigated.